Boxazomycin A and B, new antibiotics containing benzoxazole nucleus

ABSTRACT

The novel antibiotics boxazomycin A and B, which have the formula: ##STR1## wherein R is --CH 2  OH for boxazomycin A; and R is --CH 3  for boxazomycin B. These new antibiotics, which are produced by cultivating a novel strain of Pseudonocardia (ATCC 53205), inhibit growth of aerobic Gram-positive bacteria and anaerobes with enhanced activity observed in acidic medium.

FIELD OF THE INVENTION

This invention relates to novel antibiotic compounds, their productionand recovery, their use in pharmaceutical compositions, and their use intreating microbial infections. This invention also relates to a novelmicroorganism that produces said novel antibiotic compounds whencultivated.

BACKGROUND OF THE INVENTION

The present invention relates to novel antibiotic compounds designatedas boxazomycin A and B, to pharmaceutically acceptable derivativesthereof, to pharmaceutical compositions containing said compounds, to aprocess for the production of said compounds, and to a method for thetreatment of infectious diseases caused by Gram-positive bacteria.

More particularly, the process for producing boxazomycin A and B relatesto an aerobic fermentation process using a novel actinomycete strain No.G495-11 isolated from a soil sample collected in Taiwan. Themorphological, cultural and physiological characteristics and thecellular chemical compositions described hereinafter indicate thatstrain No. G495-11 is classified as a species of the genusPseudonocardia.

SUMMARY OF THE INVENTION

The new antibiotic compounds of this invention, boxazomycin A and B,have the following structures: ##STR2## wherein R is --CH₂ OH forboxazomycin A; and R is --CH₃ for boxazomycin B.

Boxazomycin A and B, which are produced by cultivating actinomycetestrain No. G495-11 (ATCC 53205), inhibit growth of aerobic Gram-positivebacteria and anaerobes with enhanced activity observed in acidic medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ¹³ C magnetic resonance spectrum of the sodium salt ofboxazomycin A in deuterium oxide.

FIG. 2 shows the proton magnetic resonance spectrum of the sodium saltof boxazomycin A in deuterium oxide.

FIG. 3 shows the infrared spectrum of boxazomycin A in the free acidform.

FIG. 4 shows the infrared spectrum of boxazomycin B in the free acidform.

DETAILED DESCRIPTION OF THE INVENTION

The novel antibiotic compounds boxazomycin A and B, named for theirbenzoxazole necleus, are fermentation products of actinomycete strainNo. G495-11, which is classified as a species of the genusPseudonocardia. A biologically pure culture of the organism has beendeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, and added to its permanent collection ofmicroorganisms as ATCC 53205. The morphological, cultural andphysiological characteristics and the cellular chemical compositions ofstrain No. G495-11 (ATCC 53205) indicate that the strain is classifiedas a species of the genus Pseudonocardia as described below:

Morphology

Strain No. G495-11 develops an abundance of long branched aerialmycelium (0.5 to 0.7 μm in width). The fragmentation of branchedsubstrate mycelium occurs after one to three days, and the resultingelements are in the form of rods of various lengths and short branchingfilaments. The following characteristics are noted: (1) the segmentationof straight hyphae, bearing long chains of cylindrical spores(0.5×1.0-3.0 μm in size) with smooth surface, (2) constriction of hyphaeto zigzag form, (3) acropetal and basipetal buddings of blastospores onrepeatedly branched aerial hyphae, occasionally with single swellingsterminally or laterally. These structures are formed both on the aerialand substrate mycelia. The Gram-stain is positive, and acid-fast stainis negative.

Cultural and Physiological Characteristics

The cultural and physiological characteristics and the carbohydrateutilization profile are shown in TABLES 1, 2 and 3, respectively. StrainNo. G495-11 grows well and forms thick white aerial mycelium on manyagar media. A yellowish, non-carotinoid, lipophilic pigment is producedin various media. Strain No. G495-11 is resistant to lysozyme, adsensitive to NaCl at 10%.

                  TABLE 1                                                         ______________________________________                                        Cultural Characteristics* of Strain No. G495-11                               ______________________________________                                        Tryptone-yeast extract                                                                     G**: No turbidity; whitish pellicle and                          broth (ISP No. 1)                                                                          colorless sediment                                                            D: None                                                          Sucrose-nitrate agar                                                                       G: Poor                                                          (Czapek's agar)                                                                            R: Yellowish white (92)                                                       A: No or scant                                                                D: None                                                          Glucose-asparagine                                                                         G: Moderate                                                      agar         R: Yellowish white (92) to brilliant                                          orange yellow (67)                                                            A: Moderate; white (263)                                                      D: Pale yellow (89)                                              Glycerol-asparagine                                                                        G: Abundant                                                      agar (ISP No. 5)                                                                           R: Deep yellow (85) to light brown (57)                                       A: Abundant; white (263) to light yellow                                      (86). Formation of blastospores                                               D: Vivid greenish yellow (97)                                    Inorganic salts-starch                                                                     G: Poor                                                          agar (ISP No. 4)                                                                           R: Yellowish white (92)                                                       A: No or scant                                                                D: None                                                          Tyrosine agar                                                                              G: Abundant                                                      (ISP No. 7)  R: Strong yellow (84) or dark olive                                           brown (96)                                                                    A: Abundant; white (263) to light                                             greenish yellow (101)                                                         D: Moderate yellow (87)                                          Nutrient agar                                                                              G: Moderate                                                                   R: Yellowish white (92)                                                       A: Scant; white (263)                                                         D: Pale yellow (89)                                              Yeast extract-malt                                                                         G: Abundant                                                      extract agar R: Brilliant yellow (83) to brownish                             (ISP No. 2)  orange (54)                                                                   A: Moderate; white (263)                                                      D: Dark orange yellow (72)                                       Oatmeal agar G: Poor                                                          (ISP No. 3)  R: Pale orange yellow (73)                                                    A: No or scant                                                                D: None                                                          Bennett's agar                                                                             G: Abundant                                                                   R: Light yellow (86) to deep orange (51)                                      A: Abundant; white (263)                                                      D: Dark yellow (88)                                              Peptone-yeast extract-                                                                     G: Moderate                                                      iron agar (ISP No. 6)                                                                      R: Strong yellow (84)                                                         A: None                                                                       D: None                                                          ______________________________________                                         *observed during incubation at 28° C. for 2 weeks.                     **abbreviation: G  Growth; R  Reverse color; A  Aerial mycelium; D            Diffusible pigment                                                            ***Color and number in parentheses follow the color standard in "Kelly, K     L. & D. B. Judd: ISCCNBS colorname charts illustrated with Centroid           Colors. US Dept. of Comm. Cir. 553, Washington, D.C., Nov., 1975".       

                                      TABLE 2                                     __________________________________________________________________________    Physiological Characteristics of Strain No. G495-11                           Test         Response     Method or medium used                               __________________________________________________________________________    Temperature for growth                                                                     Range: 12° C.-40° C.                                                         Bennett's agar                                                   Optimum: 25° C.-32° C.                                          No growth: 10° C., 42° C.                          Gelatin liquefaction                                                                       Positive (slow)                                                                            1% malt extract, 0.4%                                                         yeast extract, 0.4%                                                           glucose, 20% gelatin                                Starch hydrolysis                                                                          Negative     Starch agar plate                                   Reactions in skimmed                                                                       Peptonized and not                                                                         Difco skimmed milk                                  milk         coagulated                                                       Formation of Negative     Tyrosine agar, peptone-                             melanoid pigment          yeast extract-iron agar,                                                      and tryptone-yeast                                                            extract broth                                       Tyrosinase reaction                                                                        Negative     Arai's method*                                      Nitrate reduction                                                                          Negative     0.5% yeast extract,                                                           1% glucose, 0.5% KNO.sub.3,                                                   0.1% CaCO.sub.3.                                    pH tolerance Growth in pH 4.5-11.0                                                                      Yeast extract-malt                                                            extract agar                                        NaCl tolerance                                                                             Growth at 7% or less                                                                       Basal medium: 1% yeast                                                        extract, 2% soluble                                                           starch, 1.5% agar                                   Lysozyme tolerance                                                                         Resistant to 0.01%                                                                         Trypticase soy broth                                                          plus 1.5% agar                                      Yellow lipophilic pigment                                                                  Non-carotinoid                                                                             Pridham-Gottlieb's basal                                                      medium supplemented with                                                      xylose or galactose at                                                        5 g/L.                                              __________________________________________________________________________     *Arai, T. and Y. Mikami: Chromogenicity of Streptomyces, Appl. Microbiol.     23:402-406, 1972.                                                        

                  TABLE 3                                                         ______________________________________                                        Carbohydrate Utilization of Strain No. G495-11                                ______________________________________                                               Glycerol   +                                                                  D(-)-Arabinose                                                                           +                                                                  L(+)-Arabinose                                                                           +                                                                  D-Xylose   +                                                                  D-Ribose   +                                                                  L-Rhamnose -                                                                  D-Glucose  +                                                                  D-Galactose                                                                              +                                                                  D-Fructose +                                                                  D-Mannose  +                                                                  L(-)-Sorbose                                                                             -                                                                  Sucrose    -                                                                  Lactose    +                                                                  Cellobiose +                                                                  Melibiose  -                                                                  Trehalose  +                                                                  Raffinose  -                                                                  D(+)-Melezitose                                                                          -                                                                  Soluble starch                                                                           -                                                                  Cellulose  -                                                                  Dulcitol   -                                                                  Inositol   +                                                                  D-Mannitol +                                                                  D-Sorbitol -                                                                  Salicin    -                                                           ______________________________________                                         These results were observed after incubation at 28° C. for 2 weeks     Basal medium: PridhamGottlieb's inorganic medium, supplemented with yeast     extract at 0.2 g/L                                                            Abbreviation: +: positive utilization, -: negative utilization.          

Cellular Chemical Analyses

The following cellular components were analyzed: amino acid in purifiedcell wall; sugars in whole cell hydrolyzate; N-acyl type ofpeptidoglycan; phospholipid; and nocardomycolic acid (LCN-A). Theresults are shown in TABLE 4. Strain No. G495-11 has the composition ofcell wall Type IV-A and phospholipid Type IV. The N-acyl type ofpeptidoglycan is acetyl. Nocardo-mycolic acid is not contained.

                  TABLE 4                                                         ______________________________________                                        Cell Chemistry of Strain No. G495-11                                                       Diagnostic Component                                             ______________________________________                                        Purified cell wall                                                                           Meso-diaminopimelic acid                                       Whole cell hydrolyzate                                                                       Arabinose and galactose                                        N--Acyl type of peptido-                                                                     Acetyl                                                         glycan (glycolate test)                                                       Phospholipid   Phosphatidylethanolamine (PE),                                                phosphatidylglucosamine (Glu NU),                                             and phosphatidylcholine (PC)-trace                             Nocardo-mycolic acid                                                                         Absent                                                         ______________________________________                                    

Occurrence of Variants

The origninal culture of strain No. G495-11, which was maintained at-20° C. for 8 years, occurred in the following three tentativemorphotypes:

Type 1*: Formation of long and well-branched stable aerial mycelium

Type 2*: Formation of short rudimentary aerial mycelium

Type 3*: Lacking the ability to form aerial mycelium

A variant No. 028 of Type 3 develops true vegetative mycelium whichfragments into short filaments and rods. The surface of growth iswrinkled, granular or butyrous on the descriptive agar media. Thephysiological characteristics, such as formation of yellow pigment,profile of carbohydrate utilization and productivity of antibioticboxazomycin are well consistent with the aerial mycelium-forming strainNo. G495-11. A difference from strain No. G495-11 is observed inpositive nitrate reduction of variant No. 028.

Toxonomic Position

Based on the major characteristics mentioned above, strain No. G495-11was compared with genera Pseudonocardia, Micropolyspora,Saccharomonospora, Actinopolyspora, Saccharopolyspora and two atypicalspecies of genus Nocardia, N. autotrophica and N. orientalis.Morphologically, strain No. G495-11 forms segmented straight long hyphaewith total sporulation, blastospore-like segmented short hyphae withapical swelling, and zigzag constricting hyphae. Although the two knowspecies of genus Pseudonocardia are reported to have Type P-IIIphospholipid including phosphatidylcholine, the other majorcharacteristics indicated that strain No. G495-11 is a species of thegenus Pseudonocardia.

The following example illustrates the best mode contemplated forcarrying out the invention.

EXAMPLE Antibiotic Production

The stock culture of strain No. G495-11 was maintained on an agar slanthaving a medium composition of 1% malt extract, 0.4% glucose, 0.4% yeastextract, 0.05% CaCO₃ and 1.6% agar (pH 7.0). The culture was incubatedat 28° C. for two weeks. A well grown agar slant was used to inoculate avegetative medium consisting of 3% glucose, 3% soybean meal, 0.5% NaCland 0.1% CaCO₃. The pH of the medium was adjusted to 7.0 beforesterilization. After incubation at 28° C. for 2 days on a rotary shaker(250 rpm), 5 mL of the growth was transferred into a 500 mL Erlenmeyerflask containing 100 mL of a production medium having the samecomposition as the vegetative medium. The antibiotic activity in thefermentation broth was monitored by the paper disc-agar diffusion methodusing Bacillus subtilis PCI 219 as the test organism. The fermentationwas continued for 4 to 5 days at 28° C. on a rotary shaker and theproduction of boxazomycin reached a maximum of 50 mcg/mL.

Isolation and Purification

The harvested broth (128 L, pH 7.8) was separated to mycelial cake andsupernatant in a Sharpless centrifuge. The mycelial cake was stirred for30 minutes with methanol (30 L) and the methanolic extract evaporated invacuo to yield an aqueous concentrate. This was combined with thesupernatant obtained above, adjusted to pH 2.0 and extracted twice withethyl acetate (75 L each). After washing with water (50 L), the ethylacetate extract was shaken with dilute NaOH (35 L, pH 8.6) to transferthe activity into the aqueous solution. Upon acidification to pH 2.0with 6N hydrochloric acid, a crude yellow solid of boxazomycin complexprecipitated from the aqueous solution (24.1 g). Part of this solid (22g) was dissolved in alkaline water (350 mL, pH 10.0) and the insolublematerial was removed by filtration. Acidification of the filtrate to pH5.0 deposited a partially purified solid complex (12.2 g) whichconsisted of boxazomycin A (the major component) and B (the minorcomponent), as revealed by thin layer chromatography (TLC) (systemBT-103: CHCl₃ --MeOH--AcOH=80:20:4 in volume). The complex solid (12.2g) was dissolved in a small volume of CHCl₃ --MeOH--AcOH (80:20:4)mixture and applied on a silica gel column (6.7×50 cm) which had beenequilibrated with the same solvent. The column was eluted with theabove-mentioned solvent mixture, and the eluate was collected infractions (20 mL) and monitored by bioassay against B. subtilis PCI-219and TLC (BT-103). Fractions containing boxazomycin B (Fr. Nos. 23-27)were combined, concentrated in vacuo and lyophilized to give a paleyellow, homogeneous solid (0.19 g). Fraction Nos. 29-60 were similarlyworked up to afford pure boxazomycin A solid (2.37 g). Forcrystallization, boxazomycin A (160 mg) was dissolved in 250 mL of hotmethanol, filtered, and the filtrate was allowed to stand at roomtemperature to deposit pale yellow, fine needles of boxazomycin A freeacid (85 mg). One gram of the above solid sample was dissolved inmethanolic 0.2N NaOH (40 mL) at 70° C. and the insolubles were removedby filtration. Upon standing at room temperature, di-sodium salt ofboxazomycin A crystallized as orange columns (500 mg) which wererecrystallized from aqueous methanol (MeOH--H₂ O=10:3). The yield was305 mg.

Physico-chemical properties

Boxazomycin A and B are yellow, weakly acidic substances showing similarphysico-chemical properties. The two components are differentiated bytwo TLC systems (TABLES 5 and 6). Boxazomycin A was isolated as crystalsboth in free acid and di-sodium salt forms, while boxazomycin B has beenobtained as an amorphous powder. Boxazomycin A and B free acids arereadily soluble in alkaline water and dimethylformamide, slightlysoluble in water, methanol, ethanol and acetone, but practicallyinsoluble in ethyl acetate, chloroform, benzene and n-hexane.Boxazomycin A di-sodium salt is soluble in water. They gave a positiveresponse to ferric chloride, concentrated H₂ SO₄ and iodine reagents butwere negative in ninhydrin and anthrone tests. Molecular formulae of C₁₄H₁₂ N₄ O₅ and C₁₄ H₁₂ N₄ O₄ were assigned to boxazomycin A and B,respectively, from their mass spectral and microanalytical data. Bothcomponents exhibited a similar UV spectrum in neutral and alkalinesolutions. The physico-chemical data of boxazomycin A and B aresummarized in TABLES 5 and 6, respectively. Their IR spectra (FIGS. 3and 4) exhibited primary amide bands at 1650 and 1630 cm⁻¹, and OHand/or NH absorptions at 3350 and 3450 cm⁻¹. The ¹ H-NMR spectrum ofboxazomycin A (FIG. 2) showed the presence of one methyl (δ2.25 ppm, s),one methylene (δ4.40 ppm, s) and two aromatic protons (δ6.35 ppm, s and7.85 ppm, s). The ¹ H-NMR spectrum of boxazomycin B differed from thatof boxazomycin A only in the presence of an additional methyl group(δ2.20 ppm, s) instead of the methylene group of the latter. The ¹³C-NMR spectrum of boxazomycin A (FIG. 1) displayed 12 carbon signals,with two of them showing double intensity. Upon acetylation in pyridine,boxazomycin A afforded crystalline tetra-N-acetate (MS: molecular ion atm/z 484).

                                      TABLE 5                                     __________________________________________________________________________    Physico-Chemical Properties of Boxazomycin A                                                   Boxazomycin A                                                                 Free acid   Di--Na salt                                      __________________________________________________________________________    Nature           Pale yellow needles                                                                       Orange columns                                   M.P.             >275° C. (dec.)                                                                    >270° C. (dec.)                           Microanalysis Calcd for                                                                        C.sub.14 H.sub.12 N.sub.4 O.sub.5 1/2CH.sub.3 OH                                          C.sub.14 H.sub.10 N.sub.4 O.sub.5 Na.sub.2                                    --2H.sub.2 O                                                      C 52.41; H 4.22;                                                                          C 42.43; H 3.56;                                                  N 16.87     N 14.41; Na 11.60                                Found            C 52.80; H 4.21; N 16.59                                                                  C 41.99; H 3.24;                                                              N 14.42; Na 12.21                                EI-MS            316 (M), 299, 215                                            UV λ.sub.max nm(ε)                                                              364 (13,500)                                                 in EtOH--DMF     379.sup.sh (12,600)                                          (95:5)           420.sup.sh (6,100)                                           in 0.1N NaOH     219 (28,700)                                                                  230.sup.sh (24,800)                                                           259.sup.sh (13,100)                                                           403 (20,200)                                                 TLC (SiO.sub.2): RF                                                                            0.14                                                         n BuOH--AcOH--                                                                H.sub.2 O (3:1:1)                                                             CHCl.sub.3 --MeOH--AcOH (80:20:4)                                                              0.22                                                         __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        Physico-Chemical Properties of Boxazomycin B                                                   Boxazomycin B                                                                 Free acid                                                    ______________________________________                                        Nature             Pale yellow powder                                         M.P.               >270° C. (dec.)                                     Microanalysis      C.sub.14 H.sub.12 N.sub.4 O.sub.4                          Calcd for          C 56.00; H 4.03; N 18.66                                   Found              C 55.63; H 3.82; N 18.34                                   EI-MS              300 (M), 283, 215                                          UV λ.sub.max nm(ε)                                                                364 (14,800)                                               in EtOH--DMF       379.sup.sh (12,800)                                        (95:5)             420.sup.sh (2,200)                                         in 0.1N NaOH       219 (28,700)                                                                  230.sup.sh (24,100)                                                           259.sup.sh (11,600)                                                           403 (21,100)                                               TLC (SiO.sub.2): RF                                                                              0.44                                                       n BuOH--AcOH--H.sub.2 O                                                       (3:1:1)                                                                       CHCl.sub.3 --MeOH--AcOH                                                                          0.54                                                       (80:20:4)                                                                     ______________________________________                                    

Antimicrobial Activity and Toxicity

The minimum inhibitory concentrations (MICs) of boxazomycin A and B weredetermined by the serial agar dilution method. Nutrient agar (Eiken) wasused for aerobic bacteria, GC medium (Eiken) for fastidiuous organismsand GAM agar medium (Nissui) for anaerobic bacteria. TABLE 7 shows thein vitro antibacterial activity of boxazomycin A and B in comparisonwith kanamycin. The two components of boxazomycin showed similarantibacterial spectra, inhibiting Gram-positive aerobic andGram-positive/Gram-negative anaerobic organisms, while kanamycin wasinactive against anaerobes. The intrinsic activity of boxazomycin A wascomparable to or slightly higher than that of boxazomycin B. As shown inTABLE 8, boxazomycin A and B showed enhanced activity in acidic media,especially against anaerobic bacteria.

The in vivo efficacy of boxazomycin A was assessed in an experimentalmodel of mice infected by strains of Staphylococcus aureus andClostridium perfringens. Mice were challenged with a 100×LD₅₀ dose ofthe pathogens in a 5% suspension of hog gastric mucin (AmericanLaboratory, Omaha, Nebr.). Boxazomycin A was dissolved in saline andadministered to mice intramuscularly just before the bacterialchallenge. As shown in TABLE 9, boxazomycin A showed protective effectin mice against both infections, but its in vivo efficacy was lower thanthat expected from its in vitro activity. Boxazomycin A did not show anytoxic symptoms up to a dose of 400 mg/kg (i.m.).

                                      TABLE 7                                     __________________________________________________________________________    Antibacterial Activity of Boxazomycin A and B                                                        MIC (mcg/mL)                                                                  Boxazomycin                                            Test organisms  Test medium                                                                          A     B     Kanamycin                                  __________________________________________________________________________    Staphylococcus aureus 209P                                                                    A      0.4   0.4   0.4                                        S. aureus Smith A      0.4   0.4   0.8                                        S. aureus BX-1633.sup.b                                                                       A      0.8   0.8   0.8                                        S. aureus A20239.sup.c                                                                        A      0.4   0.8   >100                                       S. aureus A22421.sup.d                                                                        A      0.8   0.8   0.8                                        S. epidermidis A22547.sup.c                                                                   A      0.4   0.2   25                                         Streptococcus pyogenes S-23                                                                   B      3.1   12.5  25                                         S. pneumoniae IID                                                                             B      3.1   25    25                                         S. faecalis A9612                                                                             A      0.8   3.1   25                                         Micrococcus luteus PCI1001                                                                    A      0.4   0.8   3.1                                        Bacillus subtilis PCI 219                                                                     A      3.1   1.6   0.2                                        Mycobacterium smegmatis 607                                                                   A      1.6   3.1   0.8                                        M. phlei D88    A      0.8   1.6   1.6                                        M. smegmatis 607 D46.sup.c                                                                    A      1.6   3.1   >100                                       Escherichia coli NIHJ                                                                         A      >100  >100  1.6                                        Klebsiella pneumoniae D-11                                                                    A      >100  >100  0.4                                        Proteus mirabilis A9554                                                                       A      >100  >100  3.1                                        Pseudomonas aeruginosa D-113                                                                  A      >100  >100  25                                         Neisseria gonorrhoeae A15112                                                                  B      >100  >100  0.4                                        Haemophilus influenzae A9729                                                                  B      >100  >100  0.4                                        Clostridium difficile A21675.sup.e                                                            C      3.1   6.3   >50                                        C. perfringens A9635                                                                          C      3.1   12.5  >50                                        Propionibacterium acnes                                                                       C      3.1   6.3   >50                                        A21933                                                                        Peptostreptococcus                                                                            C      3.1   6.3   >50                                        anaerobius A21905                                                             B. fragilis A22534.sup.b                                                                      C      3.1   12.5  50                                         Bacteroides fragilis A22693                                                                   C      6.3   12.5  50                                         __________________________________________________________________________     .sup.a A: Nutrient agar (Eiken) B: GC medium (Eiken) C: GA agar (Nissui)      .sup.b Lactamase producer                                                     .sup.c Kanamycinresistant                                                     .sup.d Methicillinresistant                                                   .sup.e Clindamycinresistant                                              

                                      TABLE 8                                     __________________________________________________________________________    Effect of pH on Antimicrobial Activity                                                     MIC (mcg/mL)                                                                  Boxazomycin A                                                                              Boxazomycin B                                                    pH 6  pH 7   pH 6  pH 7                                          __________________________________________________________________________    B. subtilis PCI 219                                                                        0.8   6.3    0.2   1.6                                           M. luteus PCI 1001                                                                         0.1   0.4    0.2   0.8                                           S. aureus 209P                                                                             0.4   0.8    0.2   0.8                                           E. coli NIHJ >100  >100   >100  >100                                          K. pneumoniae D11                                                                          >100  >100   >100  >100                                          C. perfringens A9635                                                                       0.2   3.1    0.4   6.3                                           P. acnes A21933                                                                            0.4   6.3    0.2   6.3                                           B. fragilis A20926                                                                         0.05  3.1    0.1   3.1                                           S. necrophorus A15202                                                                      0.4   3.1    0.4   6.3                                           __________________________________________________________________________

                  TABLE 9                                                         ______________________________________                                        In Vivo Activity of Boxazomycin A                                             PD.sub.50 (mg/kg, i.m.)                                                       Test organisms    Boxazomycin A                                                                             Kanamycin                                       ______________________________________                                        Staphylococcus aureus Smith                                                                     400         1.4                                             Clostridium perfringens A9635                                                                   220         --                                              ______________________________________                                    

As shown above, boxazomycin A and B possess antimicrobial activity andare thus useful in the therapeutic treatment of mammals and otheranimals for infectious diseases caused by such microorganisms.Additionally, the compounds may be utilized for other conventionalapplications of antimicrobial agents such as disinfecting medical anddental equipment.

The present invention, therefore, provides a method for therapeuticallytreating an animal host affected by a microbial infection, which methodcomprises administering to said host an effective antimicrobial dose ofboxazomycin A or B, or a pharmaceutical composition thereof.

In another aspect, the present invention provides a pharmaceuticalcomposition which comprises an effective antimicrobial amount ofboxazomycin A or B in combination with an inert pharmaceuticallyacceptable carrier or diluent. These compositions may be made up in anypharmaceutical form appropriate for the desired use, e.g., oral orparenteral administration.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions or emulsions. They may also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, physiological saline or some other sterileinjectable medium immediately before use. Preparations for oraladministration include tablets, capsules, pills, powders, granules andliquid forms such as solutions, emulsions, suspensions, syrups andelixirs. Some of the liquid forms may also be appropriate for topicalapplication.

This invention also contemplates use of boxazomycin A and B in the formof nontoxic acid addition salts with a variety of organic and inorganicsalt-forming reagents. Such nontoxic salts are well known to thoseskilled in the art, and include acid addition salts formed with suchpharmaceutically acceptable organic or inorganic acids as sulfuric,phosphoric, hydrochloric, acetic, propionic, oleic, palmitic, citric,succinic, tartaric, glutamic, pantothenic, et. These salts are formed,isolated, purified and formulated by the methods generally employed insalt formation for antibiotics, and are employed in the same manner asthe boxazomycin A and B compounds per se.

It will be appreciated that the actual preferred amounts of boxazomycinA and B antibiotics used will vary according to the particularcomponent, the particular compositions formulated, the mode ofapplication, and the particular situs, host and disease being treated.Many factors that modify the action of the drug will be taken intoaccount by those skilled in the art, for example, age, body weight, sex,diet, time of administration, route of administration, rate ofexcretion, condition of the host, drug combinations, reactionsensitivities, and severity of the disease. Administrations can becarried out continuously or periodically within the maximum tolerateddose. Optimal application rates for a given set of conditions can beascertained by those skilled in the art using conventional dosagedetermination tests in view of the above guidelines.

We claim:
 1. The antibiotic compound boxazomycin A which has the structure: ##STR3## or a nontoxic salt thereof.
 2. The compound of claim 1 in the form of a nontoxic acid addition salt.
 3. The antibiotic compound boxazomycin B which has the structure: ##STR4## or a nontoxic acid addition salt thereof.
 4. The compound of claim 3 in the form of a nontoxic acid addition salt.
 5. A method for therapeutically treating a host animal affected by a microbial infection which comprises administering to said host animal an antimicrobially effective amount of boxazomycin A of the formula ##STR5## or a nontoxic salt thereof, or a pharmaceutical composition thereof.
 6. The method of claim 5, wherein boxazomycin A is in the form of a nontoxic acid addition salt.
 7. A method for therapeutically treating a host animal affected by a microbial infection which comprises administering to said host animal an antimicrobially effective amount of boxazomycin B of the formula ##STR6## or a nontoxic acid addition salt thereof, or a pharmaceutical composition thereof.
 8. The method of claim 7, wherein said boxazomycin B is in the form of a nontoxic acid addition salt.
 9. A pharmaceutical composition comprising an antimicrobially effective amount of boxazomycin A of the formula ##STR7## or a nontoxic salt thereof.
 10. The pharmaceutical composition of claim 9 wherein said boxazomycin A is in the form a nontoxic acid addition salt.
 11. A pharmaceutical composition comprising an antimicrobially effective amount of boxazomycin B of the formula ##STR8## or a nontoxic acid addition salt thereof.
 12. The pharmaceutical composition of claim 11, wherein boxazomycin B is in the form of a nontoxic acid addition salt. 